Compositions for affecting weight loss

ABSTRACT

Disclosed are compositions for affecting weight loss comprising a first compound and a second compound, where the first compound is an opioid antagonist and the second compound causes increased agonism of a melanocortin 3 receptor (MC3-R) or a melanocortin 4 receptor (MC4-R) compared to normal physiological conditions. Also disclosed are methods of affecting weight loss, increasing energy expenditure, increasing satiety in an individual, or suppressing the appetite of an individual, comprising identifying an individual in need thereof and treating that individual to antagonize opioid receptor activity and to enhance α-MSH activity.

RELATED APPLICATIONS

The present application claims priority to the Provisional ApplicationSer. No. 60/466,838, filed on Apr. 29, 2003, by Weber et al., andentitled “COMPOSITIONS FOR AFFECTING WEIGHT LOSS,” the entire disclosureof which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is in the field of pharmaceutical compositions andmethods for the treatment of obesity and for affecting weight loss inindividuals.

2. Description of the Related Art

Obesity is a disorder characterized by the accumulation of excess fat inthe body. Obesity has been recognized as one of the leading causes ofdisease and is emerging as a global problem. Increased instances ofcomplications such as hypertension, non-insulin dependent diabetesmellitus, arteriosclerosis, dyslipidemia, certain forms of cancer, sleepapnea, and osteoarthritis have been related to increased instances ofobesity in the general population.

Obesity has been defined in terms of body mass index (BMI). BMI iscalculated as weight (kg)/[height (m)]². According to the guidelines ofthe U.S. Centers for Disease Control and Prevention (CDC), and the WorldHealth Organization (WHO) (World Health Organization. Physical status:The use and interpretation of anthropometry. Geneva, Switzerland: WorldHealth Organization 1995. WHO Technical Report Series), for adults over20 years old, BMI falls into one of these categories: below 18.5 isconsidered underweight, 18.5-24.9 is considered normal, 25.0-29.9 isconsidered overweight, and 30.0 and above is considered obese.

Prior to 1994, obesity was generally considered a psychological problem.The discovery of the adipostatic hormone leptin in 1994 (Zhang et al.,“Positional cloning of the mouse obese gene and its human homologue,”Nature 1994; 372:425-432) brought forth the realization that, in certaincases, obesity may have a biochemical basis. A corollary to thisrealization was the idea that the treatment of obesity may be achievedby chemical approaches. Since then, a number of such chemical treatmentshave entered the market. The most famous of these attempts was theintroduction of Fen-Phen, a combination of fenfluramine and phentermine.Unfortunately, it was discovered that fenfluramine caused heart-valvecomplications, which in some cases resulted in the death of the user.Fenfluramine has since been withdrawn from the market. There has beensome limited success with other combination therapy approaches,particularly in the field of psychological eating disorders. One suchexample is Devlin, et al., Int. J. Eating Disord. 28:325-332, 2000, inwhich a combination of phentermine and fluoxetine showed some efficacyin the treatment of binge eating disorders. Of course, this disorder isan issue for only a small portion of the population.

In addition to those individuals who satisfy a strict definition ofmedical obesity, a significant portion of the adult population isoverweight. These overweight individuals would also benefit from theavailability of an effective weight-loss composition. Therefore, thereis an unmet need in the art to provide pharmaceutical compositions thatcan affect weight loss without having other adverse side effects.

SUMMARY OF THE INVENTION

Disclosed are compositions for affecting weight loss comprising a firstcompound and a second compound, where the first compound is an opioidantagonist and the second compound causes increased agonism of amelanocortin 3 receptor (MC3-R) or a melanocortin 4 receptor (MC4-R)compared to normal physiological conditions.

Also disclosed are methods of affecting weight loss, increasing energyexpenditure, increasing satiety in an individual, or suppressing theappetite of an individual, comprising identifying an individual in needthereof and treating that individual to antagonize opioid receptoractivity and to enhance α-MSH activity.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Arcuate nucleus neurons are known to be responsive to a wide array ofhormones and nutrients, including leptin, insulin, gonadal steroids, andglucose. In addition to potential transport mechanisms, peripheralsubstances may access these neurons via arcuate cell bodies in andprojections to the median eminence, a region considered to be acircumventricular organ, which lacks a blood-brain barrier. Cone et al.,“The arcuate nucleus as a conduit for diverse signals relevant to energyhomeostasis,” Int'l Journal of Obesity (2001) 25, Suppl 5, S63-S67.

Administration of exogenous leptin activates a number of differentneurons in hypothalamic and brainstem cell groups that bear leptinreceptor. Leptin-responsive neurons in the arcuate nucleus include boththose containing neuropeptide Y (NPY) and agouti-related peptide (AgRP)in the medial part of the nucleus and those containing bothpro-opiomelanocortin (POMC) and its derivatives, including α-melanocytestimulating hormone (α-MSH), as well as cocaine and amphetamine-relatedtranscript (CART). Saper et al., “The need to feed: Homeostatic andhedonic control of eating,” Neuron, 36:199-211 (2002).

The leptin-responsive POMC neurons in the arcuate nucleus are thought tocause anorexia and weigh reduction by means of the action of α-MSH onmelanocortin 3 and/or 4 receptors (MC3-R, MC4-R). The highest MC3-Rexpression level is in the hypothalamus and limbic system, whereas MC4-RmRNA is expressed in virtually all major brain regions. Some of themetabolic effects resulting from stimulation of MC4-R are decreased foodintake and an increase in energy expenditure through stimulation ofthyrotropin-releasing hormone and activation of the sympathetic nervoussystem. Targeted deletion of the MC4-R gene produces obesity,hyperphagia, hyperinsulinemia, and reduced energy expenditure. Targeteddeletion of MC3-R results in increased adiposity due to decreased energyexpenditure. Korner et al., “The emerging science of body weightregulation and its impact on obesity treatment,” J. Clin. Invest.111(5):565-570 (2003). Thus, increased concentrations of α-MSH in thecentral nervous system (CNS) increase its action on MC3-R and/or MC4-Rand result in a suppressed appetite.

POMC neurons also release β-endorphin when they release α-MSH.β-endorphin is an endogenous agonist of the 1-opioid receptors (MOP-R),found on the POMC neurons. Stimulation of MOP-R decreases the release ofα-MSH. This is a biofeedback mechanism that under normal physiologicalconditions controls the concentration of α-MSH in the CNS. Thus,blocking MOP-R by opioid antagonists will break the feedback mechanism,which results in continued secretion of α-MSH and an increase in itsconcentration in the CNS.

A second population of neurons in the arcuate nucleus tonically inhibitsthe POMC neurons. These POMC-inhibiting neurons secrete NPY, theneurotransmitter γ-aminobutyric acid (GABA), and AgRP. NPY and GABAinhibit POMC neurons, via NPY Y1 receptors and GABA receptors,respectivley. Thus, within the arcuate nucleus NPY and GABA inhibit therelease of α-MSH, and therefore are stimulators of feeding. It is knownthat leptin inhibits the release of GABA from NPY terminals synapsingonto POMC neurons, whereas ghrelin, an orexigenic peptide, stimulatesthe ghrelin receptors on NPY neurons and increase the secretion of NPYand GABA onto the POMC cells, which in turn inhibits the release ofα-MSH.

AgRP stimulates food intake in the rat through antagonism of theinteraction of α-MSH at MC4-R. Expression of the AgRP gene is suppressedby leptin.

Serotonin, also known as 5-hydroxytryptamine or 5-HT, activates the POMCneurons to secrete α-MSH. However, serotonin is taken up and removedfrom action by specific transporters so that a single serotonin moleculehas short term effects. It is known that selective serotonin re-uptakeinhibitors (SSRIs) prevent the uptake of serotonin and increase itsconcentrations in the CNS. Thus, SSRIs also increase the secretion ofα-MSH and its concentrations in the CNS.

Therefore, increased secretion of α-MSH through various mechanisms, suchas serotonin re-uptake inhibition, are among the strategies that themethods and pharmaceutical compositions of the present invention pursuein order to produce a biochemical anorexigenic effect.

The present invention provides a multi-faceted combination therapyapproach to the problem of weight loss. It addresses not just singlemolecules, messengers, or receptors, but instead acts on multiple pointsin the feeding and satiety pathway. Aspects of the present invention aredirected to increasing the concentrations of α-MSH in the CNS bystimulating the release of A-MSH, suppressing its metabolism, reducingthe antagonism of its interaction at MC3/4-R, and suppressing anyfeedback mechanisms that slow or stop its release. Aspects of thepresent invention include pharmaceutical compositions whose componentsachieve one or more of these functions. The present inventors havediscovered that a combination of two or more of the compounds disclosedherein results in a synergistic effect that affects weight loss morequickly and on a more permanent basis.

Thus, in a first aspect, the present invention is directed to acomposition for the treatment of obesity or for affecting weight losscomprising a first compound and a second compound, where the firstcompound is an opioid antagonist and the second compound causesincreased agonism of a melanocortin 3 receptor (MC3-R) or a melanocortin4 receptor (MC4-R) compared to normal physiological conditions.

In certain embodiments, the second compound causes increased activity ofthe POMC neurons, leading to greater agonism at MC3-R and/or MC4-R.

In certain embodiments the opioid antagonist antagonizes a μ-opioidreceptor (MOP-R) in a mammal. The mammal may be selected from the groupconsisting of mice, rats, rabbits, guinea pigs, dogs, cats, sheep,goats, cows, primates, such as monkeys, chimpanzees, and apes, andhumans.

In some embodiments the opioid antagonist is selected from the groupconsisting of alvimopan, norbinaltorphimine, nalmefene, naloxone,naltrexone, methylnaltrexone, and nalorphine, and pharmaceuticallyacceptable salts or prodrugs thereof.

In other embodiments, the opioid antagonist is a partial opioid agonist.Compounds of this class have some agonist activity at opioid receptors.However, because they are weak agonists, they function as de-factoantagonists. Examples of partial opioid agonists include pentacozine,buprenorphine, nalorphine, propiram, and lofexidine.

The term “pharmaceutically acceptable salt” refers to a formulation of acompound that does not cause significant irritation to an organism towhich it is administered and does not abrogate the biological activityand properties of the compound. Pharmaceutical salts can be obtained byreacting a compound of the invention with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like. Pharmaceuticalsalts can also be obtained by reacting a compound of the invention witha base to form a salt such as an ammonium salt, an alkali metal salt,such as a sodium or a potassium salt, an alkaline earth metal salt, suchas a calcium or a magnesium salt, a salt of organic bases such asdicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, and salts thereof with amino acids such as arginine,lysine, and the like.

A “prodrug” refers to an agent that is converted into the parent drug invivo. Prodrugs are often useful because, in some situations, they may beeasier to administer than the parent drug. They may, for instance, bebioavailable by oral administration whereas the parent is not. Theprodrug may also have improved solubility in pharmaceutical compositionsover the parent drug, or may demonstrate increased palatability or beeasier to formulate. An example, without limitation, of a prodrug wouldbe a compound of the present invention which is administered as an ester(the “prodrug”) to facilitate transmittal across a cell membrane wherewater solubility is detrimental to mobility but which then ismetabolically hydrolyzed to the carboxylic acid, the active entity, onceinside the cell where water-solubility is beneficial. A further exampleof a prodrug might be a short peptide (polyaminoacid) bonded to an acidgroup where the peptide is metabolized to provide the active moiety.

In certain embodiments, the second compound in the pharmaceuticalcompositions of the present invention triggers the release ofα-melanocyte stimulating hormone (α-MSH). The second compound mayincrease the extracellular serotonin concentrations in the hypothalamus.In some embodiments, the second compound is selected from the groupconsisting of a selective serotonin reuptake inhibitor (SSRI), aserotonin 2C agonist, and a serotonin 1B agonist. In furtherembodiments, the second compound is selected, e.g., from the groupconsisting of fluoxetine, fluvoxamine, sertraline, paroxetine,citalopram, escitalopram, sibutramine, duloxetine, atomoxatine, andvenlafaxine, and pharmaceutically acceptable salts or prodrugs thereof.

The terms “serotonin 1B receptor,” “serotonin 2C receptor,” “5-HT1breceptor,” and “5-HT2c receptor” refer to receptors found more commonlyin rodents. It is understood by those of skill in the art that othermammals have serotonin receptors on various neurons that are analogousin function and form to these receptors. Agonists or antagonists atthese non-rodent, preferably human, serotonin receptors are within thescope of the present invention.

In certain embodiments, the second compound suppresses the expression ofthe AgRP gene or the production or release of agouti-related protein(AgRP). In some of these embodiments, the second compound suppresses theactivity of neurons that express AgRP.

In other embodiments, the second compound suppresses the expression ofthe NPY gene or the production or release of neuropeptide Y (NPY). Insome of these embodiments, the second compound suppresses the activityof neurons that express NPY. In further embodiments, the second compoundis selected from the group consisting of NPY antagonists, ghrelinantagonists, and leptin. In certain other embodiments, the secondcompound agonizes NPY Y2 receptor.

Other embodiments of the present invention include those in which thesecond compound is selected from the group consisting of a γ-aminobutyric acid (GABA) inhibitor, a GABA receptor antagonist, and a GABAchannel antagonist. By “GABA inhibitor” it is meant a compound thatreduces the production of GABA in the cells, reduces the release of GABAfrom the cells, or reduces the activity of GABA on its receptors, eitherby preventing the binding of GABA to GABA receptors or by minimizing theeffect of such binding. The GABA inhibitor may be a 5-HT1b agonist oranother agent that inhibits the activity of NPY/AgRP/GABA neurons. Inaddition, the GABA inhibitor may suppress the expression of the AgRPgene, or the GABA inhibitor may suppress the production or release ofAgRP. It is, however, understood that a 5-HT1b agonist may inhibit theNPY/AgRP/GABA neuron (and therefore activate POMC neurons) withoutacting as an inhibitor of the GABA pathway.

In certain other embodiments the GABA inhibitor increases the expressionof the POMC gene. In some of these embodiments, the GABA inhibitorincreases the production or release of pro-opiomelanocortin (POMC)protein. In certain other of these embodiments, the GABA inhibitorincreases the activity on POMC expressing neurons. In some embodiments,the GABA inhibitor is topiramate.

In other embodiments the second compound is a dopamine reuptakeinhibitor. Phentermine is an example of a dopamine reuptake inhibitor.In certain other embodiments, the second compound is a norepinephrinereuptake inhibitor. Examples of norepinephrine reuptake inhibitorsinclude bupropion, thionisoxetine, and reboxetine. Other embodimentsinclude those in which the second compound is a dopamine agonist. Somedopamine agonists that are available on the market include cabergoline,amantadine, lisuride, pergolide, ropinirole, pramipexole, andbromocriptine. In further embodiments, the second compound is anorepinephrine releaser, for example diethylpropion, or a mixeddopamine/norepinephrine reuptake inhibitor, for example, atomoxatine.

In certain other embodiments, the second compound is a 5-HT1b agonist,such as sumatriptan, almotriptan, naratriptan, frovatriptan,rizatriptan, zomitriptan, and elitriptan.

In further embodiments, the second compound is an anticonvulsant. Theanticonvulsant may be selected from the group consisting of zonisamide,topiramate, nembutal, lorazepam, clonazepam, clorazepate, tiagabine,gabapentin, fosphenyloin, phenyloin, carbamazepine, valproate,felbamate, levetiracetam, oxcarbazepine, lamotrigine, methsuximide, andethosuxmide.

In some embodiments, the second compound is a cannabinoid receptorantagonist. Examples of this group of compounds include AM251[N-(piperidin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-1H-pyrazole-3-carboxamide],AM281[N-(morpholin-1-yl)-1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-1H-pyrazole-3-carboxamide],AM630(6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone),LY320135, and SR141716A (rimonabant), and pharmaceutically acceptablesalts or prodrugs thereof. LY320135 and SR141716A have the followingstructures.

In certain embodiments, the second compound itself may be a combinationof two or more compounds. For example, the second compound may be acombination of a dopamine reuptake inhibitor and a norepinephrinereuptake inhibitor, e.g. bupropion and mazindol. Alternatively, thesecond compound may be a combination of a SSRI and a norepinephrinereuptake inhibitor, such as sibutramine, venlafaxine, and duloxetine.

In certain embodiments, the second compound is an activator of the POMCneurons. Examples of POMC activators include Ptx1 and interleukin 1beta, (IL-1β).

In another aspect, the present invention relates to a method ofaffecting weight loss, comprising identifying an individual in needthereof and treating that individual to antagonize opioid receptoractivity and to enhance α-MSH activity.

In certain embodiments, the individual has a body mass index (BMI)greater than 25. In other embodiments, the individual has a BMI greaterthan 30. In still other embodiments, the individual has a BMI greaterthan 40. However, in some embodiments, the individual may have a BMIless than 25. In these embodiments, it may be beneficial for health orcosmetic purposes to affect weight loss, thereby reducing the BMI evenfurther.

In some embodiments, opioid receptor activity is antagonized byadministering an opioid receptor antagonist. The opioid receptorantagonist may be a MOP receptor antagonist. In some embodiments, theopioid receptor antagonist is selected from alvimopan,norbinaltorphimine, nalmefene, naloxone, naltrexone, methylnaltrexone,and nalorphine, and pharmaceutically acceptable salts or prodrugsthereof.

In some of the embodiments set forth above, α-MSH activity is enhancedby administering a compound, where the compound triggers release ofα-MSH or increases the activity of neurons that express α-MSH. In someembodiments, the compound is a selective serotonin reuptake inhibitor(SSRI) or a specific 5-HT receptor agonist. Examples of SSRIs that canbe used in the present invention include fluoxetine, fluvoxamine,sertraline, paroxetine, citalopram, escitalopram, sibutramine,duloxetine, and venlafaxine, and pharmaceutically acceptable salts orprodrugs thereof.

In other embodiments, the compound is a γ-amino butyric acid (GABA)inhibitor. The GABA inhibitor may be a 5-HT1b receptor agonist. The GABAinhibitor may suppress the expression of the AgRP gene, or it maysuppresses the production or release of AgRP. The GABA inhibitor maysuppress the expression or release of NPY. In certain embodiments, theGABA inhibitor suppresses the activity of neurons that express AgRP. Forexample, the GABA inhibitor may be topiramate,1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridinecarboxylicacid hydrochloride (NNC-711), or vigabatrin.

In certain embodiments, the method of invention set forth above ispracticed with the proviso that the individual is not suffering fromPrader-Willi syndrome or binge eating disorder. Thus, some embodimentsof the invention are to be distinguished from combination therapyinvolving SSRI anti-depressants (e.g., fluoxetine) used to treatphysiological eating disorders such as binge eating disorder orPrader-Willi syndrome. In these embodiments, the target population isthe population of individuals needing or desiring weight loss, apartfrom needing treatment for Prader-Willi syndrome or binge eatingdisorder.

Individuals suffering from depression may gain weight as a result oftheir depression. In addition, certain depressed individuals gain weightas a side effect of the depression therapy. In certain embodiments, themethod of invention set forth above is practiced with the proviso thatthe individual is not suffering from depression. In some embodiments,the individual's overweight state was not caused by treatment fordepression.

In other embodiments, the method of the invention set forth above ispracticed with the proviso that if the opioid receptor is antagonizedusing naltrexone, then release of α-MSH is not stimulated withfluoxetine. However, the combination of naltrexone with fluoxetine maybe used to affect weight loss in individuals who wish to lose weight,whether or not they are clinically categorized as obese. Theseindividuals may include those with BMI of greater than 25, or thoseindividuals with BMI of less than 25 who still wish to lose additionalweight. This particular combination may also be used for the treatmentof general obesity. In certain embodiments, the individual who wishes tolose additional weight does not suffer from binge eating disorder.

In some embodiments, the treating step of the above method comprisesadministering to the individual a first compound and a second compound,where the first compound is an opioid antagonist and the second compoundenhances α-MSH activity.

In some embodiments the first compound and the second compound areadministered more or less simultaneously. In other embodiments the firstcompound is administered prior to the second compound. In yet otherembodiments, the first compound is administered subsequent to the secondcompound.

In certain embodiments, the first compound and the second compound areadministered individually. In other embodiments, the first compound andthe second compound are covalently linked to each other such that theyform a single chemical entity. The single chemical entity is thendigested and is metabolized into two separate physiologically activechemical entities, one of which is the first compound and the other oneis the second compound.

In some embodiments, the compositions of the present invention are acombination of the following compounds:

-   -   a SSRI in combination with a dopamine reuptake inhibitor, a        dopamine/norepinephrine reuptake inhibitor, a norepinephrine        reuptake inhibitor, an opioid antagonist, a partial opioid        agonist, GABA inhibitor, a peripherally acting weight loss agent        such as metformin, or a peptide, such as PYY, PYY₃₋₃₆, or        leptin;    -   Serotonin in combination with a dopamine reuptake inhibitor, a        dopamine/norepinephrine reuptake inhibitor, an opioid        antagonist, a partial opioid agonist, or a GABA inhibitor;    -   a dopamine reuptake inhibitor in combination with a        norepinephrine reuptake inhibitor, a norepinephrine releaser, a        norepinephrine agonist, an opioid antagonist, a partial opioid        agonist, a GABA inhibitor, an adenosine compound, a cholinergic        receptor antagonist, or a peptide, such as PYY, PYY₃₋₃₆, or        leptin;    -   a dopamine/norepinephrine reuptake inhibitor in combination with        an opioid antagonist, a partial opioid agonist, a GABA        inhibitor, or a peripherally acting weight loss agent such as        metformin;    -   a dopamine agonist in combination with an opioid antagonist, a        partial opioid agonist, a GABA inhibitor, or a peptide, such as        PYY, PYY₃₋₃₆, or leptin.

Examples of norepinephrine agonists include phendimetrazine andbenzphetamine. Examples of adenosine compounds include all xanthinederivatives, such as adenosine, caffeine, theophylline, theobromine, andaminophylline. An example of acholinergic receptor antagonist isnicotine.

In another aspect, the present invention relates to a method ofincreasing satiety in an individual comprising identifying an individualin need thereof and treating that individual to antagonize opioidreceptor activity and to enhance α-MSH activity.

In some embodiments, the treating step of the above method comprisesadministering to the individual a first compound and a second compound,where the first compound is an opioid antagonist and the second compoundenhances α-MSH activity.

In some embodiments the first compound and the second compound areadministered nearly simultaneously. In other embodiments the firstcompound is administered prior to the second compound. In yet otherembodiments, the first compound is administered subsequent to the secondcompound.

In yet another aspect, the present invention relates to a method ofsuppressing the appetite of an individual comprising identifying anindividual in need thereof and treating that individual to antagonizeopioid receptor activity and to enhance α-MSH activity.

In some embodiments, the treating step of the above method comprisesadministering to the individual a first compound and a second compound,where the first compound is an opioid antagonist and the second compoundenhances α-MSH activity.

In some embodiments the first compound and the second compound areadministered nearly simultaneously. In other embodiments the firstcompound is administered prior to the second compound. In yet otherembodiments, the first compound is administered subsequent to the secondcompound.

In another aspect, the present invention relates to a method ofincreasing energy expenditure in an individual comprising identifying anindividual in need thereof and treating that individual to antagonizeopioid receptor activity and to enhance α-MSH activity.

In some embodiments, the treating step of the above method comprisesadministering to the individual a first compound and a second compound,where the first compound is an opioid antagonist and the second compoundenhances α-MSH activity.

In some embodiments the first compound and the second compound areadministered nearly simultaneously. In other embodiments the firstcompound is administered prior to the second compound. In yet otherembodiments, the first compound is administered subsequent to the secondcompound.

In certain embodiments disclosed herein, an individual is given apharmaceutical composition comprising a combination of two or morecompounds to affect weight loss. In some of these embodiments, eachcompound is a separate chemical entity. However, in other embodiments,the two compounds are joined together by a chemical linkage, such as acovalent bond, so that the two different compounds form separate partsof the same molecule. The chemical linkage is selected such that afterentry into the body, the linkage is broken, such as by enzymatic action,acid hydrolysis, base hydrolysis, or the like, and the two separatecompounds are then formed.

Thus, in another aspect, the present invention relates to syntheticroutes to novel molecules in which an opioid antagonist is linked by aflexible linker to a selective serotonin reuptake inhibitor (SSRI).

Data from previous structure-activity relationship (SAR) studies withinthe family of μ opioid antagonists may be used as a guide to determinewhich antagonists to use and the optimal position or positions on theantagonist molecules to attach the tether such that potency andselectivity of the antagonist will remain high. Similarly, SAR datawithin the family of SSRIs may be used as a guide to determine whichinhibitors to use and the optimal position or positions on theinhibitors to attach the tether such that potency and selectivity remainhigh. The tether or linker moiety is chosen from among those ofdemonstrated utility for linking bioactive molecules together. Disclosedherein are representative opioid antagonists, linkers and SSRI moleculesthat can be attached together in different combinations to formheterobivalent therapeutic molecules.

Structure-activity relationships of the opioid agonists and antagonistshave been reviewed. See for example, Zimmerman, D. M.; Leander, J. D. J.Med. Chem. 1990, 33, 895; Portoghese, P. S. J. Med. Chem. 1992, 35,1927; Carroll, F. I. J. Med. Chem. 2003, 46, 1. The opioid antagonists,nalmefene (1), naltrexone (2), naloxone (3) and naltrexamine (4) arethebaine-derived structures that share a common opiate-type template.1-Subtype selective opioid antagonists are of considerable currentinterest as agents for the treatment of obesity (Glass, M. J.;Billington, C. J.; Levine, A. S. Neuropeptides 1999, 33, 350) and CNSdisorders (Reneric, J. P.; Bouvard, M. P. CNS Drugs 1998, 10, 365).

N-Methyl and N-2-phenylethyl substituted opioids tend to show opioidagonist activity whereas N-allyl and N-cyclopropylmethyl substitutedanalogs tend to show opioid antagonist activity. Any N-attached linkermoiety will be larger than methyl. Provided that the linker moiety doesnot mimic 2-phenylethyl, such linked opioids are expected to behave asopioid antagonists. Therefore, the nitrogen atom of nalmefene andnaltrexone (and naloxone) is a suitable site for attachment of a linkermoiety. Less SAR information is available with regard to substitution atother sites on these opioids, however, attachment of the linker unit toone or the other of the carbon atoms bearing one or more hydrogen atomsremains an option.

Both nalmefene and naltrexone are potent μ-opioid antagonists. The onlystructural difference is that nalmefene has a methylene group in placeof the ketone oxygen atom in naltrexone. It is thus postulated thatsignificant changes in structure at the ketone oxygen site in naltrexonedo not significantly affect antagonist potency. Therefore, a linker maybe attached to the methylene group in nalmefene without significantreduction in antagonist potency. Carbonyl derivatives of naloxone arewell known and include symmetrical azine (═N—N═), mixed azine(Schmidhammer, H.; Kaspar, F.; Marki, A.; Borsodi, A. Helv. Chim. Acta1994, 77, 999), hydazone (Hahn, E. F.; Itzhak, Y.; Nishimura, S.;Johnson, N.; Pasternak, G. W. J. Pharm. Exper. Therapeutics 1985, 235,846-50), semicarbazone and thiosemicarbazone derivatives (Kolb, V. M.;Koman, A.; Neil, A. Pharmaceutical Res. 1985, 6, 266-71). Naloxazone,the hydrazone of naloxone, is an irreversible, selective and long actingantagonist of the μ-1 subclass of the opioid receptors (Pasternak, G.W.; Hahn, E. F. J. of Med. Chem. 1980, 23, 674-6). Certain of thederivatives are potent μ opioid antagonists while others are potentagonists.

Naltrexamine (4) has been linked by attachment of its primary aminogroup to a wide variety of other molecules producing, for example, afluorogenic opioid receptor affinity label (Le Bourdonnec, B.; ElKouhen, R.; Lunzer, M. M.; Law, P. Y.; Loh, H. H.; Portoghese, P. S.; J.Med. Chem.; 2000; 43; 2489-2492), an extensive series of nonequilibriumopioid agonists and antagonists (Sayre, L. M.; Larson, D. L.; Takemori,A. E.; Portoghese, P. S. J. Med. Chem. 1984, 27, 1325), and a series ofpotent bivalent opioid antagonists (Erez, M.; Takemori, A. E.;Portoghese, P. S. J. Med. Chem. 1982, 25, 847-849). Consequently, theprimary amino group of naltrexamine constitutes a suitable site forattachment of a linker moiety.

A limited SAR for fluoxetine (5) has been published in U.S. Pat. No.4,214,081, incorporated by reference herein in its entirety.N-Methylfluoxetine (6) shows comparable potency and selectivity to thatof fluoxetine toward inhibition of serotonin reuptake. Therefore,attachment of a linker to the nitrogen atom of fluoxetine can result inretention of the potency and selectivity of fluoxetine itself. However,the present disclosure is not limited to the fluoxetine series of SSRIs.It is envisaged that a variety of SSRI molecules such as paroxetine(Dechant, K. L.; Clissold, S. P. Drugs, 1991, 41, 225-253) or one or theother of the bivalent SSRIs described by Kozikowski et al. (Tamiz, A.P.; Zhang, J.; Zhang, M.; Wang, C. Z.; Johnson, K. M.; Kozikowski, A. P.J. Am. Chem. Soc. 2000, 122, 5393-5394; Tamiz, A. P.; Bandyopadhyay, B.C.; Zhang, J.; Flippen-Anderson, J. L.; Zhang, M.; Wang, C. Z.; Johnson,K. M.; Tella, S.; Kozikowski, A. P. J. Med. Chem. 2001, 44, 1615-1622)may also be utilized to construct the heterobivalent therapeuticmolecules of this invention.

Examples of linkers reported in the scientific literature includemethylene (CH₂)_(n) linkers (Hussey, S. L.; Muddana, S. S.; Peterson, B.R.; J. Am. Chem. Soc. 2003; 125; 3692-3693; Tamiz, A. P.; Bandyopadhyay,B. C.; Zhang, J.; Flippen-Anderson, J. L.; Zhang, M.; Wang, C. Z;Johnson, K. M.; Tellar, S.; Kozikowski, A. P. J. Med. Chem. 2001, 44,1615-1622), oligo ethyleneoxy O(—CH₂CH₂O—)_(n) units used to linknaltrexamine to other opioids, glycine oligomers of the formula—NH—(COCH₂NH)_(n)COCH₂CH₂CO—(NHCH₂CO)_(n)NH— used to link opioidantagonists and agonists together ((a) Portoghese, P. S.; Ronsisvalle,G.; Larson, D. L.; Yim, C. B.; Sayre, L. M.; Takemori, A. E. Life Sci.1982, 31, 1283-1286. (b) Portoghese, P. S.; Larson, D. L.; Sayre, L. M.;Yim, C. B.; Ronsisvalle, G.; Tam, S. W.; Takemori, A. E. J. Med. Chem.1986, 29, 1855-1861), hydrophilic diamines used to link opioid peptidestogether (Stepinski, J.; Zajaczkowski, I.; Kazem-Bek, D.; Temeriusz, A.;Lipkowski, A. W.; Tam, S. W. Internat. J. of Peptide & Protein Res.1991, 38, 588-92), rigid double stranded DNA spacers (Paar, J. M.;Harris, N. T.; Holowka, D.; Baird, B. J. Immunol. 2002, 169, 856-864)and the biodegradable linker poly (L-lactic acid) (Klok, H.-A.; Hwang,J. J.; Iyer, S. N.; Stupp, S. I. Macromolecules 2002, 35, 746-759). Theattachment of the tether to the antagonist can result in the antagonistachieving a favorable binding orientation. The linker itself may or maynot be biodegradable. The linker may take the form of a prodrug and betunable for optimal release kinetics of the linked drugs. The linker maybe either conformationally flexible throughout its entire length or elsea segment of the tether may be designed to be conformationallyrestricted (Portoghese, P. S.; Ronsisvalle, G.; Larson, D. L.; Takemori,A. E. J. Med. Chem. 1986, 29, 1650-1653).

In Scheme 1 below, naltrexone (2) is used in the linking reaction. As aconsequence of the Wittig reaction, a double bond replaces the carbonylgroup in naltrexone. The net result is fluoxetine linked with a flexiblemethylene linker to a nalmefene molecule by way of the nalmefene doublebond.

Reductive amination of fluoxetine with an ω-bromoaldehyde such as11-bromoundecanal 6 (n=9) gives bromoamine 7 (n=9), best stored as thehydrobromide salt to prevent an unwanted slow macrocyclization sidereaction by way of attack of the free amino group on the carbon bearingthe bromine atom. Reaction of 7 with triphenylphosphine gives theintermediate phosphonium salt, which upon rection with butyllithiumgenerates the corresponding ylid 8 (n=9). A Wittig reaction between 8and the ketone group of naltrexone (2) gives the linked molecule 9containing a fluoxetine unit coupled to what is now a nalmefene unit.The expected mixture of cis, trans isomers about the newly introduceddouble bond is separable by standard chromatographic techniques. Ifracemic fluoxetine is used, then a mixture of two optically activediastereomers of 9 will be produced owing to the fact that a singleenantiomer 2 of naltrexone was used. Chemists skilled in the art willrecognize that the (CH₂)₉ linker may be varied in length and/or containsubstituents by beginning with a different bromoaldehyde. Thus,pharmacological properties may be optimized. Molecule 9 is stable underphysiological conditions. Opioid antagonist activity will be due to thecovalently linked nalmefene unit and not due to free nalmefene releasedas a result of some cleavage reaction. Similarly, SSRI activity will bedue to the covalently linked fluoxetine unit and not due to freefluoxetine released as a result of some cleavage reaction.

An analogous reaction sequence may be used in which the bromoaldehyde isderived from an oligo ethylene glycol as shown in Scheme 2 below. Forexample, tetraethylene glycol (10 n=2) is converted into bromide 11(n=2), which is then oxidized under Swern conditions to aldehyde 12(n=2). Substitution of aldehyde 12 for aldehyde 6 in Scheme 1 will givea series of irreversibly linked molecules in which the linker is morehydrophilic than that in molecules 9. Generation of the ylid in theoligo ethylene glycol series and the subsequent Wittig reaction isperformed at reduced temperature to avoid β-elimination of the alkoxygroup. If racemic fluoxetine is used, then a mixture of two opticallyactive diastereomers of 13 will be produced owing to the fact that asingle enantiomer 2 of naltrexone was used. Chemists skilled in the artwill recognize that the (OCH₂CH₂)_(n) linker may be varied in length bybeginning with a different bromoaldehyde 12. Thus, pharmacologicalproperties may be optimized. Molecule 13 is stable under physiologicalconditions.

In Scheme 3, another linking method beginning with tetraethylene glycolis illustrated as an example of a variety of oligo ethylene glycols thatmay be used. Adapting the chemistry of Sashiwa et al. (Sashiwa, H.;Shigemasa, Y.; Roy, R. Macromolecules 2000, 33, 6913), tetraethyleneglycol may be converted into acetal 14 (n=2) and subsequently intoaldehyde 15. Reductive amination of fluoxetine with aldehyde 15 givesthe fluoxetine derivative 16. Reduction of azide 16 to amine 17 and thenreductive amination with naltrexone gives molecule 18 in which afluoxetine unit is linked irreversibly by a flexible oligo ethyleneoxyunit to β-naltrexamine (after separation of the α and β isomers). Ifracemic fluoxetine is used, then a mixture of two optically activediastereomers of 18 will be produced owing to the fact that a singleenantiomer 2 of naltrexone was used. Chemists skilled in the art willrecognize that the (OCH₂CH₂)_(n) linker may be varied in length bybeginning with a different oligo ethylene glycol 10. Thus,pharmacological properties may be optimized. Molecule 18 should bestable under physiological conditions.

Scheme 4 illustrates a synthetic route to fluoxetine linked to nalmefeneby way of the N-cyclopropyl group of nalmefene. The readily availablet-butyldimethylsilyl protected noroxymorphone (19) is synthesized frommorphine (Ninan, A.; Sainsbury, M. Tetrahedron 1992, 48, 6709-16), andthen subjected to a reductive amination reaction with the commerciallyavailable cyclopropanecarboxaldehyde 20 (Aldrich, largely trans) givingester 21. Wittig methyleneation gives ester 22, which is hydrolyzed togive acid 23. Activation of acid 23 with an appropriate carbodiimide andthen N-acylation of fluoxetine derivative 17 (Scheme 3) gives 25,deprotection of which with Bu₄NF gives the novel molecule 26. Chemistsskilled in the art will recognize that the (OCH₂CH₂)_(n) linker may bevaried in length by beginning with a different aldehyde azide 15 in thesynthesis of 17. Thus, pharmacological properties may be optimized.Molecule 26 should be stable under physiological conditions.

Alternatively, ester 22 may be reduced to aldehyde 24 using DIBAL at−78° C. Reductive amination of aldehyde 24 with amine 17 gives molecule27 after removal of the TBDMS protecting group. Chemists skilled in theart will recognize that the (OCH₂CH₂)_(n) linker may be varied in lengthby beginning with a different aldehyde azide 15 in the synthesis of 17.Thus, pharmacological properties may be optimized. Molecule 27 should bestable under physiological conditions.

If the Wittig methyleneation step is omitted in the above sequence, thenan analog of 26, namely ketone 28, is formed in which the methylenegroup of 26 is replaced by a carbonyl group. The result is a naltrexoneunit linked to a fluoxetine unit by way of a flexible, hydrophilic(CH₂CH₂O)_(n) linker in the form of compound 28. Chemists skilled in theart will recognize that the (OCH₂CH₂)_(n) linker may be varied in lengthby beginning with a different aldehyde azide 15 in the synthesis of 17.Thus, pharmacological properties may be optimized. Molecule 28 is stableunder physiological conditions.

Scheme 5 illustrates how fluoxetine may be linked to β-naltrexamineusing a combination of linkers, namely the flexible glycine-basedlinkers 29 exploited by Portoghese et al. and the oligo ethylene glycollinkers used in the schemes above. Thus carboxyl activation of 29 with asuitable carbodiimide followed by monocondensation with β-naltrexaminegives amide 30. Reactivation of 30 followed by condensation with amine17 (Scheme 3) gives molecule 31. Portoghese reports that symmetricalamides derived from linker 29 and β-naltrexamine are effective μ-opioidreceptor antagonists. Chemists skilled in the art will recognize thatthe —NH—(COCH₂NH)_(n−1)COCH₂CH₂CO—(NHCH₂CO)_(n)NH— linker may be variedin length by beginning with a different glycine-based linking unit 29 inthe synthesis of 30. Thus, pharmacological properties may be optimized.Molecule 31 is stable under physiological conditions.

Reaction of bromide 7 (Scheme 1) with Mg in dry THF will give Grignardreagent 32, reaction of which with the carbonyl group of naltrexonegives adduct 33 after separation of the two diastereomers produced atthe newly created chiral center. Adduct 33 contains a fluoxetine segmentlinked to a N-cyclopropylmethyl-normorphine unit by way of a flexiblemethylene linker. Chemists skilled in the art will recognize that the(CH₂)₉ linker may be varied in length by beginning with a differentbromoaldehyde for the synthesis of bromide 7. Thus, pharmacologicalproperties may be optimized. Molecule 33 is stable under physiologicalconditions.

Throughout the above schemes, one should be able to employN-desmethylfluoxetine (34), or any other derivative of fluoxetine, inplace of fluoxetine. The resulting linked fluoxetine unit is identicalto that of fluoxetine itself except that the methyl group of fluoxetineis replaced by a longer chain that is part of the linker. When necessarydue to the use of strongly basic reagents or when chemoselectivitytoward a primary amino group elsewhere in the molecule is required, onemay protect the intermediate fluoxetine secondary amino group by use ofthe N-[2-(trimethylsilyl)ethoxy]methyl (SEM) group (Zeng, Z.; Zimmerman,S. C. Tetrahedron Lett. 1988, 29, 5123) as illustrated in Scheme 7.

In another aspect, the invention relates to a pharmaceutical compositioncomprising a combination of an opioid antagonist and a compound thatcauses increased agonism of a melanocortin 3 receptor (MC3-R) or amelanocortin 4 receptor (MC4-R) compared to normal physiologicalconditions, as described above, or comprising a linked molecule, asdescribed herein, and a physiologically acceptable carrier, diluent, orexcipient, or a combination thereof.

The term “pharmaceutical composition” refers to a mixture of a compoundof the invention with other chemical components, such as diluents orcarriers. The pharmaceutical composition facilitates administration ofthe compound to an organism. Multiple techniques of administering acompound exist in the art including, but not limited to, oral,injection, aerosol, parenteral, and topical administration.Pharmaceutical compositions can also be obtained by reacting compoundswith inorganic or organic acids such as hydrochloric acid, hydrobromicacid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and thelike.

The term “carrier” defines a chemical compound that facilitates theincorporation of a compound into cells or tissues. For example dimethylsulfoxide (DMSO) is a commonly utilized carrier as it facilitates theuptake of many organic compounds into the cells or tissues of anorganism.

The term “diluent” defines chemical compounds diluted in water that willdissolve the compound of interest as well as stabilize the biologicallyactive form of the compound. Salts dissolved in buffered solutions areutilized as diluents in the art. One commonly used buffered solution isphosphate buffered saline because it mimics the salt conditions of humanblood. Since buffer salts can control the pH of a solution at lowconcentrations, a buffered diluent rarely modifies the biologicalactivity of a compound.

The term “physiologically acceptable” defines a carrier or diluent thatdoes not abrogate the biological activity and properties of thecompound.

The pharmaceutical compositions described herein can be administered toa human patient per se, or in pharmaceutical compositions where they aremixed with other active ingredients, as in combination therapy, orsuitable carriers or excipient(s). Techniques for formulation andadministration of the compounds of the instant application may be foundin “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton,Pa., 18th edition, 1990.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intravenous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intraperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directly inthe renal or cardiac area, often in a depot or sustained releaseformulation. Furthermore, one may administer the drug in a targeted drugdelivery system, for example, in a liposome coated with atissue-specific antibody. The liposomes will be targeted to and taken upselectively by the organ.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or tabletting processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen. Any of the well-knowntechniques, carriers, and excipients may be used as suitable and asunderstood in the art; e.g., in Remington's Pharmaceutical Sciences,above.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained by mixing one or more solid excipient withpharmaceutical combination of the invention, optionally grinding theresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

A pharmaceutical carrier for the hydrophobic compounds of the inventionis a cosolvent system comprising benzyl alcohol, a nonpolar surfactant,a water-miscible organic polymer, and an aqueous phase. A commoncosolvent system used is the VPD co-solvent system, which is a solutionof 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate80™, and 65% w/v polyethylene glycol 300, made up to volume in absoluteethanol. Naturally, the proportions of a co-solvent system may be variedconsiderably without destroying its solubility and toxicitycharacteristics. Furthermore, the identity of the co-solvent componentsmay be varied: for example, other low-toxicity nonpolar surfactants maybe used instead of POLYSORBATE 80™; the fraction size of polyethyleneglycol may be varied; other biocompatible polymers may replacepolyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars orpolysaccharides may substitute for dextrose.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are well knownexamples of delivery vehicles or carriers for hydrophobic drugs. Certainorganic solvents such as dimethylsulfoxide also may be employed,although usually at the cost of greater toxicity. Additionally, thecompounds may be delivered using a sustained-release system, such assemipermeable matrices of solid hydrophobic polymers containing thetherapeutic agent. Various sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of the therapeuticreagent, additional strategies for protein stabilization may beemployed.

Many of the compounds used in the pharmaceutical combinations of theinvention may be provided as salts with pharmaceutically compatiblecounterions. Pharmaceutically compatible salts may be formed with manyacids, including but not limited to hydrochloric, sulfuric, acetic,lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble inaqueous or other protonic solvents than are the corresponding free acidor base forms.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions where the active ingredients are contained in anamount effective to achieve its intended purpose. More specifically, atherapeutically effective amount means an amount of compound effectiveto prevent, alleviate or ameliorate symptoms of disease or prolong thesurvival of the subject being treated. Determination of atherapeutically effective amount is well within the capability of thoseskilled in the art, especially in light of the detailed disclosureprovided herein.

The exact formulation, route of administration and dosage for thepharmaceutical compositions of the present invention can be chosen bythe individual physician in view of the patient's condition. (See e.g.,Fingl et al. 1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1p. 1). Typically, the dose range of the composition administered to thepatient can be from about 0.5 to 1000 mg/kg of the patient's bodyweight. The dosage may be a single one or a series of two or more givenin the course of one or more days, as is needed by the patient. Notethat for almost all of the specific compounds mentioned in the presentdisclosure, human dosages for treatment of at least some condition havebeen established. Thus, in most instances, the present invention willuse those same dosages, or dosages that are between about 0.1% and 500%,more preferably between about 25% and 250% of the established humandosage. Where no human dosage is established, as will be the case fornewly-discovered pharmaceutical compounds, a suitable human dosage canbe inferred from ED₅₀ or ID₅₀ values, or other appropriate valuesderived from in vitro or in vivo studies, as qualified by toxicitystudies and efficacy studies in animals.

Although the exact dosage will be determined on a drug-by-drug basis, inmost cases, some generalizations regarding the dosage can be made. Thedaily dosage regimen for an adult human patient may be, for example, anoral dose of between 0.1 mg and 500 mg of each ingredient, preferablybetween 1 mg and 250 mg, e.g. 5 to 200 mg or an intravenous,subcutaneous, or intramuscular dose of each ingredient between 0.01 mgand 100 mg, preferably between 0.1 mg and 60 mg, e.g. 1 to 40 mg of eachingredient of the pharmaceutical compositions of the present inventionor a pharmaceutically acceptable salt thereof calculated as the freebase, the composition being administered 1 to 4 times per day.Alternatively the compositions of the invention may be administered bycontinuous intravenous infusion, preferably at a dose of each ingredientup to 400 mg per day. Thus, the total daily dosage by oraladministration of each ingredient will typically be in the range 1 to2000 mg and the total daily dosage by parenteral administration willtypically be in the range 0.1 to 400 mg. Suitably the compounds will beadministered for a period of continuous therapy, for example for a weekor more, or for months or years.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain themodulating effects, or minimal effective concentration (MEC). The MECwill vary for each compound but can be estimated from in vitro data.Dosages necessary to achieve the MEC will depend on individualcharacteristics and route of administration. However, HPLC assays orbioassays can be used to determine plasma concentrations.

Dosage intervals can also be determined using MEC value. Compositionsshould be administered using a regimen which maintains plasma levelsabove the MEC for 10-90% of the time, preferably between 30-90% and mostpreferably between 50-90%.

In cases of local administration or selective uptake, the effectivelocal concentration of the drug may not be related to plasmaconcentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. The pack or dispensermay also be accompanied with a notice associated with the container inform prescribed by a governmental agency regulating the manufacture,use, or sale of pharmaceuticals, which notice is reflective of approvalby the agency of the form of the drug for human or veterinaryadministration. Such notice, for example, may be the labeling approvedby the U.S. Food and Drug Administration for prescription drugs, or theapproved product insert. Compositions comprising a compound of theinvention formulated in a compatible pharmaceutical carrier may also beprepared, placed in an appropriate container, and labeled for treatmentof an indicated condition.

It will be understood by those of skill in the art that numerous andvarious modifications can be made without departing from the spirit ofthe present invention. Therefore, it should be clearly understood thatthe forms of the present invention are illustrative only and are notintended to limit the scope of the present invention.

Some Embodiments of the Invention

Some of the embodiments of the present invention are as follows:

In the first embodiment, the invention relates to a composition foraffecting weight loss comprising a first compound and a second compound,wherein said first compound is an opioid antagonist and said secondcompound causes increased agonism of a melanocortin 3 receptor (MC3-R)or a melanocortin 4 receptor (MC4-R) compared to normal physiologicalconditions.

In the second embodiment, the invention relates to the composition ofthe first embodiment, wherein said opioid antagonist antagonizes anopioid receptor in a mammal.

In the third embodiment, the invention relates to the composition of thesecond embodiment, wherein said opioid receptor is selected from aμ-opioid receptor (MOP-R), a η-opioid receptor, and a δ-opioid receptor.

In the fourth embodiment, the invention relates to the composition ofthe second embodiment, wherein said opioid antagonist antagonizes aμ-opioid receptor (MOP-R) in a mammal.

In the fifth embodiment, the invention relates to the composition of thefirst embodiment, wherein said opioid antagonist is selected from thegroup consisting of alvimopan, norbinaltorphimine, nalmefene, naloxone,naltrexone, methylnaltrexone, and nalorphine, and pharmaceuticallyacceptable salts or prodrugs thereof.

In the sixth embodiment, the invention relates to the composition of thefirst embodiment, wherein said opioid antagonist is a partial opioidagonist.

In the seventh embodiment, the invention relates to the composition ofthe sixth embodiment, wherein said partial opioid agonist is selectedfrom the group consisting of pentacozine, buprenorphine, nalorphine,propiram, and lofexidine.

In the eighth embodiment, the invention relates to the composition ofthe first embodiment, wherein said second compound triggers the releaseof α-melanocyte stimulating hormone (α-MSH).

In the ninth embodiment, the invention relates to the composition of theeighth embodiment, wherein said second compound increases theextracellular serotonin concentrations in the hypothalamus.

In the tenth embodiment, the invention relates to the composition of theninth embodiment, wherein said second compound is selected from thegroup consisting of a selective serotonin reuptake inhibitor (SSRI), aserotonin 2C agonist, and a serotonin 1B agonist.

In the eleventh embodiment, the invention relates to the composition ofthe tenth embodiment, wherein said second compound is selected from thegroup consisting of fluoxetine, fluvoxamine, sertraline, paroxetine,citalopram, escitalopram, sibutramine, duloxetine, and venlafaxine, andpharmaceutically acceptable salts or prodrugs thereof.

In the twelfth embodiment, the invention relates to the composition ofthe first embodiment, wherein said second compound suppresses theexpression of the AgRP gene or the production or release ofagouti-related protein (AgRP).

In the thirteenth embodiment, the invention relates to the compositionof the first embodiment, wherein said second compound suppresses theactivity of neurons that express AgRP.

In the fourteenth embodiment, the invention relates to the compositionof the first embodiment, wherein said second compound suppresses theexpression of the NPY gene or the production or release of neuropeptideY (NPY).

In the fifteenth embodiment, the invention relates to the composition ofthe first embodiment, wherein said second compound suppresses theactivity of neurons that express NPY.

In the sixteenth embodiment, the invention relates to the composition ofthe first embodiment, wherein said second compound is selected from thegroup consisting of NPY Y1 receptor antagonists, ghrelin antagonists,and leptin.

In the seventeenth embodiment, the invention relates to the compositionof the first embodiment, wherein said second compound agonizes NPY Y2receptor.

In the eighteenth embodiment, the invention relates to the compositionof the first embodiment, wherein said second compound is selected fromthe group consisting of a γ-amino butyric acid (GABA) inhibitor, a GABAreceptor antagonist, and a GABA channel antagonist.

In the nineteenth embodiment, the invention relates to the compositionof the eighteenth embodiment, wherein said GABA inhibitor is a 5-HT1bagonist, which may be selected from sumatriptan, almotriptan,naratriptan, frovatriptan, rizatriptan, zomitriptan, and elitriptan.

In the twentieth embodiment, the invention relates to the composition ofthe eighteenth embodiment, wherein said GABA inhibitor suppresses theexpression of the AgRP gene.

In the twenty first embodiment, the invention relates to the compositionof the eighteenth embodiment, wherein said GABA inhibitor suppresses theproduction or release of AgRP.

In the twenty second embodiment, the invention relates to thecomposition of the eighteenth embodiment, wherein said GABA inhibitorincreases the expression of the POMC gene.

In the twenty third embodiment, the invention relates to the compositionof the eighteenth embodiment, wherein said GABA inhibitor increases theproduction or release of α-MSH from pro-opiomelanocortin (POMC) neurons.

In the twenty fourth embodiment, the invention relates to thecomposition of the eighteenth embodiment, wherein said GABA inhibitorincreases the activity of POMC expressing neurons.

In the twenty fifth embodiment, the invention relates to the compositionof the eighteenth embodiment, wherein the GABA inhibitor is topiramate.

In the twenty sixth embodiment, the invention relates to the compositionof the first embodiment, wherein said second compound is a dopaminereuptake inhibitor.

In the twenty seventh embodiment, the invention relates to thecomposition of the twenty sixth embodiment, wherein said dopaminereuptake inhibitor is phentermine.

In the twenty eighth embodiment, the invention relates to thecomposition of the first embodiment, wherein said second compound is anorepinephrine reuptake inhibitor.

In the twenty ninth embodiment, the invention relates to the compositionof the twenty eighth embodiment, wherein said norepinephrine reuptakeinhibitor is selected from bupropion, thionisoxetine, and reboxetine.

In the thirtieth embodiment, the invention relates to the composition ofthe first embodiment, wherein said second compound is a dopamineagonist.

In the thirty first embodiment, the invention relates to the compositionof the thirtieth embodiment, wherein said dopamine agonist is selectedfrom the group consisting of cabergoline, amantadine, lisuride,pergolide, ropinirole, pramipexole, and bromocriptine.

In the thirty second embodiment, the invention relates to thecomposition of the first embodiment, wherein said second compound is anorepinephrine releaser.

In the thirty third embodiment, the invention relates to the compositionof the thirty second embodiment, wherein said norepinephrine releaser isdiethylpropion.

In the thirty fourth embodiment, the invention relates to thecomposition of the first embodiment, wherein said second compound is acombination of a dopamine reuptake inhibitor and a norepinephrinereuptake inhibitor.

In the thirty fifth embodiment, the invention relates to the compositionof the thirty fourth embodiment, wherein said second compound isselected from bupropion and mazindol.

In the thirty sixth embodiment, the invention relates to the compositionof the first embodiment, wherein said second compound is a combinationof a SSRI and a norepinephrine reuptake inhibitor.

In the thirty seventh embodiment, the invention relates to thecomposition of the thirty sixth embodiment, wherein said second compoundis selected from sibutramine, venlafaxine, and duloxetine.

In the thirty eighth embodiment, the invention relates to thecomposition of the first embodiment, wherein said first compound isnaltrexone and said second compound is fluoxetine.

In the thirty ninth embodiment, the invention relates to the compositionof the thirty eighth embodiment, wherein the naltrexone is in atime-release formulation whereas the fluoxetine is in an immediaterelease formulation.

In the fortieth embodiment, the invention relates to a method ofaffecting weight loss, comprising identifying an individual in needthereof and treating that individual to antagonize opioid receptoractivity and to enhance α-MSH activity.

In the forty first embodiment, the invention relates to the method ofthe fortieth embodiment, wherein said individual has a body mass indexgreater than 25.

In the forty second embodiment, the invention relates to the method ofthe fortieth embodiment, wherein opioid receptor activity is antagonizedby administering an opioid receptor antagonist.

In the forty third embodiment, the invention relates to the method ofthe forty second embodiment, wherein the opioid receptor antagonist is aMOP receptor antagonist.

In the forty fourth embodiment, the invention relates to the method ofthe fortieth embodiment, wherein the opioid receptor antagonist isselected from alvimopan, norbinaltorphimine, nalmefene, naloxone,naltrexone, methylnaltrexone, and nalorphine, and pharmaceuticallyacceptable salts or prodrugs thereof.

In the forty fifth embodiment, the invention relates to the method ofthe forty second embodiment, wherein said opioid receptor antagonist isa partial opioid agonist.

In the forty sixth embodiment, the invention relates to the method ofthe forty fifth embodiment, wherein said partial opioid agonist isselected from the group consisting of pentacozine, buprenorphine,nalorphine, propiram, and lofexidine.

In the forty seventh embodiment, the invention relates to the method ofthe fortieth embodiment through the forty fifth embodiment, whereinα-MSH activity is enhanced by administering a compound, wherein saidcompound triggers release of α-MSH or increases the activity of neuronsthat express α-MSH.

In the forty eighth embodiment, the invention relates to the method ofthe forty seventh embodiment, wherein said compound is a selectiveserotonin reuptake inhibitor (SSRI) or a specific 5-HT receptor agonist.

In the forty ninth embodiment, the invention relates to the method ofthe forty eighth embodiment, wherein said 5-HT receptor is selected from5-HT1b receptor and 5-HT2c receptor.

In the fiftieth embodiment, the invention relates to the method of theforty eighth embodiment, wherein said SSRI is selected from fluoxetine,fluvoxamine, sertraline, paroxetine, citalopram, escitalopram,sibutramine, duloxetine, and venlafaxine, and pharmaceuticallyacceptable salts or prodrugs thereof.

In the fifty first embodiment, the invention relates to the method ofthe forty seventh embodiment, wherein said compound is a γ-amino butyricacid (GABA) inhibitor.

In the fifty second embodiment, the invention relates to the method ofthe fifty first embodiment, wherein said GABA inhibitor is a 5-HT1breceptor agonist.

In the fifty third embodiment, the invention relates to the method ofthe fifty first embodiment, wherein said GABA inhibitor suppresses theexpression of the AgRP gene.

In the fifty fourth embodiment, the invention relates to the method ofthe fifty first embodiment, wherein said GABA inhibitor suppresses theproduction or release of AgRP.

In the fifty fifth embodiment, the invention relates to the method ofthe forty eighth embodiment, wherein said 5-HT agonists inhibits theNPY/AgRP/GABA neurons.

In the fifty sixth embodiment, the invention relates to the method ofthe fifty first embodiment, wherein said GABA inhibitor suppresses theactivity of neurons that express AgRP.

In the fifty seventh embodiment, the invention relates to the method ofthe fifty first embodiment, wherein said GABA inhibitor is topiramate.

In the fifty eighth embodiment, the invention relates to the method ofthe forty seventh embodiment, wherein said compound is selected from thegroup consisting of a dopamine reuptake inhibitor, a norepinephrinereuptake inhibitor, a dopamine agonist, a norepinephrine releaser, acombination of a dopamine reuptake inhibitor and a norepinephrinereuptake inhibitor, and a combination of a SSRI and a norepinephrinereuptake inhibitor.

In the fifty ninth embodiment, the invention relates to the method ofthe fifty eighth embodiment, wherein said compound is not phentermine.

In the sixtieth embodiment, the invention relates to the method of thefortieth embodiment, with the proviso that the individual is notsuffering from Prader-Willi syndrome.

In the sixty first embodiment, the invention relates to the method ofthe fortieth embodiment, with the proviso that if the opioid receptor isantagonized using naltrexone, then release of α-MSH is not stimulatedwith fluoxetine.

In the sixty second embodiment, the invention relates to the method ofthe fortieth embodiment, wherein said treating step comprisesadministering to said individual a first compound and a second compound,wherein said first compound is an opioid antagonist and said secondcompound enhances α-MSH activity.

In the sixty third embodiment, the invention relates to the method ofthe sixty second embodiment, wherein said first compound and said secondcompound are administered nearly simultaneously.

In the sixty fourth embodiment, the invention relates to the method ofthe sixty third embodiment, wherein said first compound is administeredprior to said second compound.

In the sixty fifth embodiment, the invention relates to the method ofthe sixty fourth embodiment, wherein said first compound is administeredsubsequent to said second compound.

In the sixty sixth embodiment, the invention relates to a method ofincreasing satiety in an individual comprising identifying an individualin need thereof and treating that individual to antagonize opioidreceptor activity and to enhance α-MSH activity.

In the sixty seventh embodiment, the invention relates to the method ofthe sixty sixth embodiment, wherein said treating step comprisesadministering to said individual a first compound and a second compound,wherein said first compound is an opioid antagonist and said secondcompound enhances α-MSH activity.

In the sixty eighth embodiment, the invention relates to the method ofthe sixty seventh embodiment, wherein said first compound and saidsecond compound are administered nearly simultaneously.

In the sixty ninth embodiment, the invention relates to the method ofthe sixty seventh embodiment, wherein said first compound isadministered prior to said second compound.

In the seventieth embodiment, the invention relates to the method of thesixty seventh embodiment, wherein said first compound is administeredsubsequent to said second compound.

In the seventy first embodiment, the invention relates to a method ofincreasing energy expenditure in an individual comprising identifying anindividual in need thereof and treating that individual to antagonizeopioid receptor activity and to enhance α-MSH activity.

In the seventy second embodiment, the invention relates to the method ofthe seventy first embodiment, wherein said treating step comprisesadministering to said individual a first compound and a second compound,wherein said first compound is an opioid antagonist and said secondcompound enhances α-MSH activity.

In the seventy third embodiment, the invention relates to the method ofthe seventy second embodiment, wherein said first compound and saidsecond compound are administered nearly simultaneously.

In the seventy fourth embodiment, the invention relates to the method ofthe seventy second embodiment, wherein said first compound isadministered prior to said second compound.

In the seventy fifth embodiment, the invention relates to the method ofthe seventy second embodiment, wherein said first compound isadministered subsequent to said second compound.

In the seventy sixth embodiment, the invention relates to a method ofsuppressing the appetite of an individual comprising identifying anindividual in need thereof and treating that individual to antagonizeopioid receptor activity and to enhance α-MSH activity.

In the seventy seventh embodiment, the invention relates to the methodof the seventy sixth embodiment, wherein said treating step comprisesadministering to said individual a first compound and a second compound,wherein said first compound is an opioid antagonist and said secondcompound enhances α-MSH activity.

In the seventy eighth embodiment, the invention relates to the method ofthe seventy seventh embodiment, wherein said first compound and saidsecond compound are administered nearly simultaneously.

In the seventy ninth embodiment, the invention relates to the method ofthe seventy seventh embodiment, wherein said first compound isadministered prior to said second compound.

In the eightieth embodiment, the invention relates to the method of theseventy seventh embodiment, wherein said first compound is administeredsubsequent to said second compound.

In the eighty first embodiment, the invention relates to a method ofaffecting weight loss in an individual comprising identifying anindividual in need thereof and treating that individual with acombination of naltrexone and fluoxetine,

provided that the individual does not suffer from Prader-Willi syndromeor binge eating disorder.

In the eighty second embodiment, the invention relates to the method ofthe eighty first embodiment, wherein the individual has a BMI greaterthan 30.

In the eighty third embodiment, the invention relates to the method ofthe eighty first embodiment, wherein the individual has a BMI greaterthan 25.

In the eighty fourth embodiment, the invention relates to the method ofthe eighty first embodiment, wherein the naltrexone is in a time-releaseformulation whereas the fluoxetine is in an immediate releaseformulation.

In the eighty fifth embodiment, the invention relates to the method ofthe eighty fourth embodiment, wherein the plasma concentration level ofboth naltrexone and fluoxetine follow a similar concentration profile.

In the eighty sixth embodiment, the invention relates to the method ofthe eighty fourth embodiment, wherein the naltrexone and the fluoxetineare administered substantially simultaneously.

In the eighty seventh embodiment, the invention relates to the method ofthe eighty fourth embodiment, wherein the naltrexone is administeredprior to the fluoxetine.

In the eighty eighth embodiment, the invention relates to the method ofthe eighty fourth embodiment, wherein the naltrexone is administeredsubsequent to the fluoxetine.

EXAMPLES

The examples below are non-limiting and are merely representative ofvarious aspects of the invention.

Example 1 Combination of Fluoxetine and Naltrexone

Individuals having a BMI of greater than 25 are identified. Eachindividual is instructed to take one 20 mg tablet of fluoxetine(PROZAC®) on a daily basis, in addition to one 50 mg tablet ofnaltrexone on a daily basis.

The individuals are monitored for a period of months. It is recommendedthat the dosage be adjusted so that each individual loses weight at arate of 10% of initial weight every 6 months. However, the rate of weighloss for each individual may be adjusted by the treating physician basedon the individual's particular needs.

If the initial dosage is not effective, then the fluoxetine dosage canbe increased by 20 mg per day, though never exceeding 80 mg total perday. If the initial dosage results in a more rapid weight loss than theabove rate, the dosage of each of fluoxetine or naltrexone can bereduced.

Fluoxetine has a physiological half life of about 9 hours, whereas thatof naltrexone is about 1.5 hours. Thus, in some cases, it is beneficialto administer one dose of fluoxetine per day in conjunction with two orthree or more doses of naltrexone throughout the day. Naltrexone mayalso be in a time-release formulation where the dose is administeredonce a day, but naltrexone gradually enters the blood stream throughoutthe day, or in the course of a 12 hour period.

Example 2 Combination of Fluoxetine and Nalmefene

Individuals having a BMI of greater than 25 are identified. Eachindividual is instructed to take one 20 mg tablet of fluoxetine(PROZAC®) on a daily basis. In addition, each individual is injectedwith 1 mL of a solution of 100 μg of nalmefene in 1 mL of saline,intravenously, intramuscularly, or subcutaneously.

The individuals are monitored for a period of months. It is recommendedthat the dosage be adjusted so that each individual loses weight at arate of 10% of initial weight every 6 months. However, the rate of weighloss for each individual may be adjusted by the treating physician basedon the individual's particular needs.

If the initial dosage is not effective, then the fluoxetine dosage canbe increased by 20 mg per day, though never exceeding 80 mg total perday. In addition, the dosage of nalmefene may be increased up to 2 mL ofa solution of 1 mg of nalmefene in 1 mL of saline. If the initial dosageresults in a more rapid weight loss than the above rate, the dosage ofeach of fluoxetine or nalmefene can be reduced.

Example 3 Combination of Fluoxetine and Naloxone

Individuals having a BMI of greater than 25 are identified. Eachindividual is instructed to take one 20 mg tablet of fluoxetine(PROZAC®) on a daily basis. In addition, each individual is injectedwith 1 mL of a solution of 400 μg of naloxone in 1 mL of saline,intravenously, intramuscularly, or subcutaneously.

The individuals are monitored for a period of months. It is recommendedthat the dosage be adjusted so that each individual loses weight at arate of 10% of initial weight every 6 months. However, the rate of weighloss for each individual may be adjusted by the treating physician basedon the individual's particular needs.

If the initial dosage is not effective, then the fluoxetine dosage canbe increased by 20 mg per day, though never exceeding 80 mg total perday. If the initial dosage results in a more rapid weight loss than theabove rate, the dosage of each of fluoxetine or nalmefene can bereduced.

Example 4 Combination of Opioid Antagonist and Sibutramine

Individuals having a BMI of greater than 25 are identified. Eachindividual is instructed to take nalmefene, naltrexone, or naloxone inthe dosage set forth in Examples 1-3. In addition, each individual isinstructed to take 10 mg of sibutramine orally once a day.

The individuals are monitored for a period of months. It is recommendedthat the dosage be adjusted so that each individual loses weight at arate of 10% of initial weight every 6 months. However, the rate of weighloss for each individual may be adjusted by the treating physician basedon the individual's particular needs.

If the initial dosage is not effective, then the sibutramine dosage canbe increased 15 mg per day. Dosages of sibutramine in excess of 15 mgper day are not recommended. If the initial dosage results in a morerapid weight loss than the above rate, the dosage of each ofsibutramine, nalmefene, naltrexone, or naloxone can be reduced.

Example 5 Combination of Opioid Antagonist and Bupropion

Individuals having a BMI of greater than 25 are identified. Eachindividual is instructed to take nalmefene, naltrexone, or naloxone inthe dosage set forth in Examples 1-3. In addition, each individual isinstructed to take bupropion. The usual adult does is 300 mg per day,given three times daily. Dosing should begin at 200 mg per day, given as100 mg twice daily. Based on clinical response, this dose may beincreased to 300 mg per day, given as 100 mg three times daily. Nosingle dose is to exceed 150 mg.

The individuals are monitored for a period of months. It is recommendedthat the dosage be adjusted so that each individual loses weight at arate of 10% of initial weight every 6 months. However, the rate of weighloss for each individual may be adjusted by the treating physician basedon the individual's particular needs.

Example 6 Combination of Opioid Antagonist and Phentermine

Individuals having a BMI of greater than 25 are identified. Eachindividual is instructed to take nalmefene, naltrexone, or naloxone inthe dosage set forth in Examples 1-3. In addition, each individual isinstructed to take 37.5 mg of phentermine orally once a day.

The individuals are monitored for a period of months. It is recommendedthat the dosage be adjusted so that each individual loses weight at arate of 10% of initial weight every 6 months. However, the rate of weighloss for each individual may be adjusted by the treating physician basedon the individual's particular needs.

Example 7 Combinations with Naltrexone

In a multicenter, randomized, blinded, placebo-controlled clinical trialwith 6 groups, the following drug combinations are tested:

-   -   Group 1: Fluoxetine 60 mg po QD plus Naltrexone 50 mg po QD    -   Group 2: Fluoxetine 60 mg po QD plus N-placebo po QD    -   Group 3: Bupropion-SR 150 mg po BID plus Naltrexone 50 mg po QD    -   Group 4: Bupropion-SR 150 mg po BID plus N-placebo po QD    -   Group 5: P-placebo po BID plus Naltrexone 50 mg po QD    -   Group 6: P-placebo po BID plus N-placebo po QD

In any of the above groups, the dosage of fluoxetine may be in the rangebetween 6 mg and 60 mg, for example, 6 mg, 10 mg, 12 mg, 18 mg, 20 mg,24 mg, 30 mg, 36 mg, 40 mg, 42 mg, 45 mg, 48 mg, 54 mg, and 60 mg.Bupropion may be administered in doses in the range between 30 mg and300 mg, for example, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg,100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg,190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 260 mg, 270 mg,280 mg, 290 mg, and 300 mg. Naltrexone may be administered in doses inthe range between 5 mg and 50 mg, for example, 5 mg, 10 mg, 15 mg, 20mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, and 50 mg.

Subjects are evaluated as out-patients during this study. All subjectsin this trial receive diet instruction, behavior modification advice andinstruction to increase their activity, a regimen shown to give weightloss. Subjects are randomized to receive study drugs in variouscombinations.

Subjects in groups 5 and 6 cross-over to treatment with fluoxetine plusnaltrexone or bupropion SR plus naltrexone after week 16 for theextension treatment period which provide additional data on safety ofthe combination therapies.

The primary endpoint is percent and absolute change from baseline inbody weight at 16 weeks. Secondary endpoints include weight loss at 24,36, and 48 weeks, number and proportion of subjects who achieve at leasta 5% weight loss and a 10% weight loss (responder analysis), changes inobesity-associated cardiovascular risk factors (total cholesterol, LDLcholesterol, HDL cholesterol, triglycerides, glucose and insulin) andwaist circumference, and safety and tolerability. Adverse events,laboratory parameters, vital signs, and the Hospital Anxiety andDepression (HAD) Scale are used to monitor safety and tolerability.

Example 8 Dose-Response Experiments

Seventy, four week old, male C57/B16J⁻ mice (Jackson Laboratory), 22-30g were sham injected daily with 0.1 mL 0.9% saline (pH 7.4) for 1 weekprior to the experiments. Animals were weighed and randomized to 1 of 7weight-matched dose groups (0, 1.5, 3, 5.5, 10, 18, and 30 mg/kg;n=10/group for fluoxetine; 0, 1.5, 3, 5.5, 10, 18, and 30 mg/kg;n=3/group for naltrexone) the day before experiments began. Food wasremoved between 4:30-5:30 pm the day before the experiment. Animalsreceived a 0.3 mL bolus (fluoxetine) or 0.1 mL bolus (naltrexone)intraperitoneal injection between 9-10:30 am, and food was providedimmediately following injection. 3 animals/group received injections oneach testing day (i.e., 3 runs of 3/group; 1 run of 1/group). Food wasweighed 1, 2, 4, 8, and 24 h post-injection. Cumulative food intake±SEMwas calculated and analyzed using Prizm. Doses were log transformed andfit to a sigmoidal curve, food intake was expressed as a proportion ofthe food intake in saline treated animals. From the curve, the EC₅₀ ateach time point for each drug was determined.

Similar procedures as described above were followed using AM251 andnalmefene, fluvoxamine and nalmefene, and bupropion and naltrexone.

The results are set forth in the table below:

Hour 1 Hour 2 Hour 4 Hour 8 Hour 8 SEM SEM SEM SEM SEM MEAN (±) MEAN (±)MEAN (±) MEAN (±) MEAN (±) Saline 1.00 0.0690 1.00 0.062 1.00 0.047 1.000.052 1.00 0.042 AM 251 0.97 0.086 0.85 0.060 0.88 0.057 0.90 0.036 0.990.054 Fluvoxamine 0.77 0.058 0.85 0.056 0.95 0.044 0.91 0.034 0.92 0.054Nalmefene 0.0083 0.0062 0.11 0.061 0.57 0.11 0.81 0.068 0.98 0.027 AM251 + 0.010 0.010 0.075 0.055 0.30 0.1 0.62 0.042 0.90 0.021 NalmefeneFluvoxamine + 0.0041 0.0041 0.019 0.012 0.42 0.087 0.79 0.026 0.99 0.031Nalmefene Bupropion 0.32 0.044 0.64 0.049 0.97 0.048 0.96 0.036 0.990.020 Naltrexone 0.41 0.040 0.77 0.060 0.99 0.062 1.1 0.048 0.98 0.030Naltrexone + 0.042 0.0068 0.34 0.10 0.89 0.055 0.97 0.044 0.95 0.028Bupropion Naltrexone 0.30 0.10 0.56 0.050 0.83 0.10 0.98 0.19 1.01 0.22Fluoxetine 0.36 0.030 0.57 0.13 0.68 0.15 0.76 0.21 1.05 0.22Naltrexone + 0.070 0.030 0.26 0.060 0.72 0.11 0.95 0.18 1.04 0.26Fluoxetine

Example 9 Electrophysiology Data

To test the hypothesis that drugs selectively activate POMC neurons, weused a strain of transgenic mice expressing green fluorescent protein(EGFP, Clontech), under the transcriptional control of mouse Pomcgenomic sequences that include a region located between −13 kb and −2 kbrequired for accurate neuronal expression Bright green fluorescence (509nm) was seen in the two CNS regions where POMC is produced: the ARC andthe nucleus of the solitary tract. Under ultraviolet (450-480 nm)excitation, POMC neurons were clearly distinguished from adjacent,non-fluorescent neurons visualized under infrared optics.

200 μm thick coronal slices were cut from the ARC of four-week old malePOMC-EGFP mice. Slices were maintained in Krebs solution (NaCl (126 mM),KCl (2.5 mM), MgCl₂ 91.2 mM), CaCl₂.2H₂O (2.4 mM), NaH₂PO₄.H₂O (1.2 mM),NaHCO₃ (21.4 mM), glucose (11.1 mM)) at 35° C. and saturated with 95% O₂and 5% CO₂ for 1 hr prior to recordings. Recordings were made in Krebsat 35° C. Slices were visualized on an Axioskop FS2 plus (Zeiss) throughstandard infra red optics and using epifluorescence through a FITC(longpass) filter set. POMC-EGFP neurons in hypothalamic slices had aresting membrane potential of −40 to −45 mV and exhibited frequentspontaneous action potentials. Cell-attached recordings were made fromfluorescent neurons using an Axopatch 200B amplifier (Axon Instruments)and Clampex 8 (Axon Instruments). Action potentials frequencies weredetermined using an event detection program (Mini Analysis; SynaptosoftInc., Decatur, Ga.). Drugs were applied to the bath for 3 min.

Data were analyzed by determining the average firing rate for 500 secprior to drug addition, and analyzing treatments relative to thisfrequency (that is, firing rates were normalized to the pre-treatmentfrequency). The ratio's listed for the combinations are the ratio of theeffect of naltrexone in combination with the POMC activator, relative tonaltrexone alone (that is the extra effectiveness that naltrexoneconferred to the POMC activator). Also listed are the mean effects ofthe drugs alone.

Fenfluramine   2X increase (n = 6) Fenfluramine + Naltrexone 5.2X (n =8) Fluoxetine   3X (n = 1) Fluoxetine + Naltrexone 1.2X (n = 1) Dopamine 11X (n = 9) Dopamine + Naltrexone 1.5X (n = 3)

Naltrexone alone has a potent (7×) but variable effect. many cells didnot respond to naltrexone alone, but gave a significant response tocombination treatment. Heisler et al. (Science 297(5581):609-11 (2002))show that fenfluramine alone causes a 200% effect.

The results are set forth in the table below:

Drug Dose Effect (%) Drug Dose Effect (%) Ratio Naltrexone 1 μM 29650Naltrexone + 1 μM + 20 μM 15080 0.51 Fenfluramine Naltrexone 1 μM 2200Naltrexone + 1 μM + 20 μM 11440 520 Fenfluramine Naltrexone 1 μM 2500Naltrexone + 1 μM + 20 μM 856 0.34 Fenfluramine Naltrexone 1 μM 417Naltrexone + 1 μM + 20 μM 5700 13.67 Fenfluramine Naltrexone 1 μM 177Naltrexone + 1 μM + 20 μM 430 2.43 Fenfluramine Naltrexone 1 μM 200Naltrexone + 1 μM + 20 μM 2933 14.67 Fenfluramine Naltrexone 1 μM 700Naltrexone + 1 μM + 20 μM Fenfluramine Naltrexone 1 μM 900 Naltrexone +1 μM + 20 μM 1831 2.03 Fenfluramine Naltrexone 1 μM 2273 Naltrexone + 1μM + 20 μM Fenfluramine Naltrexone 1 μM 300 Naltrexone + 1 μM + 20 μM920 3.07 Fenfluramine

1. A composition for affecting weight loss comprising: (a) a sustainedrelease formulation of bupropion or a pharmaceutically acceptable saltthereof in an amount effective to induce weight loss in an individual;and (b) a sustained release formulation of naltrexone or apharmaceutically acceptable salt thereof in an amount effective toenhance the weight loss effect of the bupropion or salt thereof; whereinsaid composition is in a single oral dosage form fixed combination. 2.The composition of claim 1, wherein said composition comprises about 5mg to about 50 mg of naltrexone or a pharmaceutically acceptable saltthereof.
 3. The composition of claim 1, wherein said compositioncomprises about 30 mg to about 500 mg of bupropion or a pharmaceuticallyacceptable salt thereof.
 4. The composition of claim 1, wherein saidcomposition comprises about 5 mg to about 50 mg of naltrexone or apharmaceutically acceptable salt thereof, and about 30 mg to about 500mg of bupropion or a pharmaceutically acceptable salt thereof.
 5. Apharmaceutical composition for affecting weight loss comprising: (a) asustained release formulation of bupropion or a pharmaceuticallyacceptable salt thereof in an amount effective to induce weight loss inan individual; (b) a sustained release formulation of naltrexone or apharmaceutically acceptable salt thereof in an amount effective toenhance the weight-reducing effect of the bupropion or salt thereof; anda pharmaceutically acceptable excipient, diluent, or carrier, whereinsaid composition is formulated into a single oral fixed combinationdosage form.
 6. The pharmaceutical composition of claim 5, wherein saidcomposition comprises about 5 mg to about 50 mg of naltrexone or apharmaceutically acceptable salt thereof.
 7. The pharmaceuticalcomposition of claim 5, wherein said composition comprises about 30 mgto about 500 mg of bupropion or a pharmaceutically acceptable saltthereof.
 8. The pharmaceutical composition of claim 5, wherein saidcomposition comprises about 5 mg to about 50 mg of naltrexone or apharmaceutically acceptable salt thereof, and about 30 mg to about 500mg of bupropion or a pharmaceutically acceptable salt thereof.